PROJECT SUMMARY/ABSTRACT It was reported that in 2009 alone, 32% of all deaths in the US resulted from cardiovascular disease, including acute coronary syndrome, stroke/transient ischemic attack, and peripheral artery disease. Underlying these pathologies is increased platelet activity. Platelets play an essential role in hemostasis and thrombosis. Following vascular and tissue damage, platelets rapidly adhere to the site of injury and secrete granular contents that recruit and activate additional platelets, culminating in the formation of thrombi through the interaction between integrin ?IIb?3 and fibrinogen. Because of the essential role of integrin ?IIb?3 in platelet thrombus formation, antagonists of the integrin have been developed. However, direct inhibition of ligand binding to integrin ?IIb?3 could impair hemostasis and increase the risk of bleeding. Thus, many efforts have been put forward to develop inhibitors blocking integrin ?IIb?3 function or signaling. Numerous studies have now characterized the role of thiol-disulfide bond exchange as a regulatory mechanism for integrin ?IIb?3. Using real-time intravital microscopy, we and others demonstrated that the isomerase activity of cell surface protein disulfide isomerase (PDI), a prototypical thiol isomerase, plays a critical role in regulating integrin ?3 activation and thrombus formation at the site of vascular injury, thereby suggesting PDI to be a novel therapeutic target for the treatment of thrombosis. However, blocking extracellular PDI compromised hemostatic function in mice as assessed by tail bleeding times. These results warrant the need for further research into how extracellular PDI activity is regulated following vascular injury. In the endoplasmic reticulum (ER), PDI catalyzes thiol-disulfide oxidation, reduction, and isomerization during protein folding and ER oxidoreductin 1? (ERO1?) is the key protein responsible for accepting the electrons from reduced PDI to oxidize PDI, regenerating its activity. In this comprehensive proposal, we will test the hypothesis that platelet surface ERO1? modulates the ligand-binding function of integrin ?IIb?3 by controlling PDI activity and facilitates platelet aggregation during thrombosis. The proposed studies will provide mechanistic insight into an innovative approach to downregulate the ligand-binding function of integrin ?IIb?3 by inhibition of the ERO1?-PDI signaling axis and may lead to the development of novel therapeutic strategies.